Widespread mermithid nematode parasitism of Cretaceous insects

Mermithid nematodes are obligate invertebrate parasites dating back to the Early Cretaceous. Their fossil record is sparse, especially before the Cenozoic, thus little is known about their early host associations. This study reports 16 new mermithids associated with their insect hosts from mid-Cretaceous Kachin amber, 12 of which include previously unknown hosts. These fossils indicate that mermithid parasitism of invertebrates was already widespread and played an important role in the mid-Cretaceous terrestrial ecosystem. Remarkably, three hosts (bristletails, barklice, and perforissid planthoppers) were previously unknown to be parasitized by mermithids both past and present. Furthermore, our study shows that in contrast to their Cenozoic counterparts, Cretaceous nematodes including mermithids are more abundant in non-holometabolous insects. This result suggests that nematodes had not completely exploited the dominant Holometabola as their hosts until the Cenozoic. This study reveals what appears to be a vanished history of nematodes that parasitized Cretaceous insects.


Introduction
Nematodes (roundworms), a group of non-segmented worm-like invertebrates, are some of the most abundant animals on earth in terms of individuals (Lorenzen, 1994;Poinar, 2011). They are distributed worldwide in almost all habitats and play key roles in ecosystems by linking soil food webs, influencing plant growth and facilitating nutrient cycling (Yeates et al., 2009;van den Hoogen et al., 2019;Zhang et al., 2020). The earliest known definite nematode fossil occurs in the Lower Devonian Rhynie Chert inside the cortex cells of an early land plant and has been considered to be a plant parasite , but unconfirmed nematode-like fossils and trace fossils may date back to the Precambrian (Poinar, 1979;Parry et al., 2017;

Systematic palaeontology
Fossil nematodes can be attributed to the family Mermithidae based mainly on their relatively large size, coiled posture, and morphological comparison with extant mermithids (body shape, length, diameter, tail structure, etc.) (Poinar, 2011). They can also be distinguished from nematomorphs due to the lack of small elevations of irregular areas (areoles) on their epicuticles (Poinar, 2001b). Due to palaeontological inability to adequately detect all biological adult characters, it is impossible to place in or refer fossil mermithid nematodes to any natural extant genus. This is why fossil collective genera have been erected under the same guidelines as recent collective genera for difficult nematodes. The importance of placing nematode species in collective genera is to underpin or establish the time, place and hosts of these parasitic lineages. For Cretaceous Mermithidae not assignable to any previously known genus or lacking biologically preferred diagnostic characters, the collective genus Cretacimermis was erected (but invalidly, see remarks for Cretacimermis below) (Poinar, 2001b). Putative hosts were determined by noting nematodes emerging from their bodies or completely emerged nematodes adjacent to potential hosts, especially if there is physical evidence that a particular insect was parasitized.

Cretacimermis directa
Remarks. There is no distinct wound on this cricket's body, but the nematode is adjacent to it, and there is no other insect nearby. Therefore, it is most likely that the mermithid had just emerged from the host. Remarks. The abdomen of the first perforissid planthopper is empty, which probably contained the developing nematode. The abdomen of the second perforissid planthopper is broken, which is consistent with an emerging mermithid.      Description. Body incomplete, tanned, partially transparent ( Figure 6I); cuticle with areas of shrinkage ridges; head missing, tail rounded ( Figure 6J); length 9.6 mm; greatest width 111 µm; a=87.

Cretacimermis manicapsoci
Remarks. The nematode is adjacent to the host and the empty abdomen indicates the area that contained the developing parasite. Etymology. The species epithet is derived from the type genus of Cecidomyiidae. Type host. Gall midge of the family Cecidomyiidae (Diptera: Culicomorpha) ( Figure 7A). Material. Holotype. Kachin amber, cabochon, 7×4×3 mm, weight 0.1 g, specimen no. NIGP201871. Diagnosis. Mermithid nematode parasitizing cecidomyiid midge from mid-Cretaceous Kachin amber. Description. Body grayish with white areas; cuticle smooth, lacking cross fibres; body with dark trophosome; head missing, tail obscured; length at least 1.5 mm; greatest width 73 µm; a=at least 21 ( Figure 7B and C).
Remarks. The mermithid is preserved in the process of emerging from the host's body.

Discussion
The sixteen new mermithids associated with their insect hosts described above include 10 insectmermithid associations. The hosts of nine species were previously unrecorded, which triples the diversity of Cretaceous Mermithidae (from 4 to 13 species). In today's ecosystems, mermithids have been reported from a variety of arthropods, in a range of environments, and often infecting large percentages of host populations and causing mass mortality (Poinar, 1975;Poinar, 1979;Petersen, 1985). Despite their abundance in extant terrestrial ecosystems, mermithids are rare in the fossil record as they are not readily preserved as fossils. Twenty-two fossil mermithid species have been described from the Cenozoic with their hosts (Supplementary file 1: Table S1), mainly from Eocene Baltic amber (11 species) and Miocene Dominican amber (9 species), but only four pre-Cenozoic species associated with only two insect orders have previously been recorded (Poinar and Buckley, 2006;Poinar and Sarto i Monteys, 2008;Poinar, 2011;Poinar, 2017). However, according to our new records, nine insect orders are now known to have been infested by mermithid nematodes in Kachin amber and this number is even higher than that of Baltic amber (~45 Ma) and Dominican amber (~18 Ma) (six and three insect orders, respectively), despite a much longer time spent searching for nematodes in the latter two amber deposits (Poinar, 2011). Together with previously described mermithids in Kachin amber (Poinar, 2001b;Grimaldi et al., 2002;Poinar and Buckley, 2006;Poinar, 2011;Poinar, 2017), our results suggest that mermithid parasitism of insects was actually widespread during the mid-Cretaceous (Figure 8). Mermithids species are usually characterized by strong host specificity, they are specific to a single species or to one or two families of insects, and are almost always lethal to their hosts (Stoffolano, 1973;Petersen, 1985), thus our study indicates that the widespread mermithid parasitism probably already played an important role in regulating the population of insects in Cretaceous terrestrial ecosystems.
Our study provides new information on fossil host-parasite associations, including three previously unknown host-mermithid associations and first fossil records of four host associations. One is Cretacimermis incredibilis sp. nov., which has completely exited from a bristletail (Archaeognatha). Its tail end  is still adjacent to an exit wound on the host (Figure 2D), indicating a true parasitic association. There are no previous extant or extinct records of nematodes attacking bristletails (Poinar, 1975;Poinar, 2011). A second new mermithid-host association is barklice (Psocodea) with three different specimens parasitized by mermithids. No barklice are parasitized by mermithids today (Poinar, 1975), but our specimens imply that such relationship might have been quite common in the mid-Cretaceous. Two members of the extinct planthopper family Perforissidae were also parasitized by mermithids, thus providing the oldest record of mermithid parasitism of planthoppers. The mermithid Heydenius brownii parasitized achiliid planthoppers in Baltic amber (Poinar, 2001a) and this association also occurs in extant planthoppers (Choo et al., 1989;Helden, 2008). Furthermore, our findings are the first fossil records of mermithids parasitizing dragonflies (Odonata), earwigs (Dermaptera), crickets (Orthoptera) and cockroaches (Blattodea), four host associations predicted from extant records (Poinar, 1975).
Nematode body fossils are scarce and mainly known from amber (De Baets et al., 2021b), sometimes together with their hosts (Poinar, 2011). To explore the evolution of nematode-host relationship, we compiled nematode-host records in the three best-studied amber biotas (mid-Cretaceous Kachin amber, Eocene Baltic amber and Miocene Dominican amber; Supplementary file 1: Table S1).
Our results indicate that not only the mermithids, but also the nematodes as a whole, experienced a certain degree of host transition between the Cretaceous and Cenozoic (Figure 9). We cannot fully exclude the possibility of collection bias, but its influence is probably low because Kachin amber has been extensively studied in the last two decades and its biota has already become the most diverse known amber biota; moreover, holometabolous insects are much more diverse in the collections than non-holometabolous ones (1296 vs 465 species : Ross, 2023). It is therefore unlikely that holometabolous insects are underrepresented among the known hosts of mermithids. Among the insect hosts of mermithids preserved in Kachin amber, only one of the nine orders (Diptera) is holometabolous (i.e. insects with 'complete' metamorphosis), whilst it is four out of six (Hymenoptera, Trichoptera, Lepidoptera and Diptera) in Baltic amber and all three insect host orders (Hymenoptera, Coleoptera and Diptera) are holometabolous in Dominican amber. The situation is similar when referring to the amount of nematode parasitism ( Table 1). In Kachin amber, only about 40% of the hosts (in total, not only insects) are holometabolous, while this percentage increases to 80% in Baltic and Dominican amber and this result is acceptable when uncertainty is considered ( Figure 9B). Diptera are the most common hosts of nematodes from all three amber biotas; also, the oldest fossil animal that was found to host a mermithid is a dipteran from Early Cretaceous Lebanese amber (Poinar et al., 1994). This is probably because most dipteran larvae develop in moist or aquatic environments that are particularly suitable habitats for nematodes (Poinar, 2011). It is evident that Holometabola are the most important hosts of extant mermithids as well as all invertebrate-parasitizing nematodes (Poinar, 1975) and this hexapod subgroup dominated the insect fauna during the Cretaceous (Labandeira and Sepkoski, 1993;Labandeira, 2005;Sohn et al., 2015;Peters et al., 2017;Zhang et al., 2018;Thomas et al., 2020;Wang et al., 2022). Our study suggests that, except for Diptera, nematodes had not completely exploited Holometabola as hosts in the mid-Cretaceous. This suggests that nonholometabolous insects (i.e. insects without 'complete' metamorphosis) were more available as hosts in the mid-Cretaceous and the widespread association between nematodes and Holometabola might have formed later.
Finally, discovering these nematodes in mid-Cretaceous Kachin amber brings new opportunities to study the evolution of parasitism through the medium of amber. Amber is a unique form of fossilization (Hsieh and Plotnick, 2020). Although amber is patchily distributed in space and time, it is still especially suitable for investigating the evolution of terrestrial parasites associated with arthropods due to preservation potential (De Baets and Littlewood, 2015;Leung, 2017;De Baets et al., 2021a;De Baets et al., 2021b;Leung, 2021;Poinar, 2021). The high diversity of mermithid nematodes    during the mid-Cretaceous as shown here provides a glimpse into the structure of ancient parasitic nematode-host associations and their evolution over the past 100 million years.

Provenance and deposition
The specimens described here are from the Cretaceous deposits in the Hukawng Valley located southwest of Maingkhwan in Kachin State (26°20' N, 96°36' E) in Myanmar (Thu and Zaw, 2017). Radiometric U-Pb zircon dating determined the age to be 98.79±0.62 Ma (Shi et al., 2012), a date consistent with an ammonite trapped in the amber (Yu et al., 2019).
Fourteen specimens (NIGP201868-201881) are deposited in the NIGPAS, and two specimens (LYD-MD-NG001, 002) are deposited in Linyi University. The fossils were collected in full compliance with the laws of Myanmar and China (work on this manuscript began in early 2016). To avoid any confusion and misunderstanding, all authors declare that to their knowledge, the fossils reported in this study were not involved in armed conflict and ethnic strife in Myanmar, and were acquired prior to 2017. All specimens are permanently deposited in well-established, public museums, in full compliance with the International Code of Zoological Nomenclature and the Statement of the International Palaeoentomological Society (International Commission on Zoological Nomenclature, 1999;Szwedo et al., 2020).

Optical photomicrography
Observations were performed using a Zeiss Stemi 508 microscope. The photographs were taken with a Zeiss Stereo Discovery V16 microscope system in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, and measurements were taken using Zen software. Photomicrographic composites of 10-150 individual focal planes were digitally stacked using the software HeliconFocus 6.7.1 for a better illustration of 3D structures. Photographs were adjusted using Adobe Lightroom Classic and line drawings were prepared using CorelDraw 2019 graphic software.